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Paranaguá-Vezozzo et al. Hepatoma Res 2018;4:11 I http://dx.doi.org/10.20517/2394-5079.2018.17 Page 3 of 11

in 12 (63.1%) of the 19 biopsies performed. The remaining seven cases were included based on the progressive
increase of nodule size; with consequent better definition by imaging methods (5 cases) and/or increased
AFP level (2 cases).

HCC diagnosis was made with imaging in 15 patients (48.3%) and histology in 12 patients (38.8%). A
combination of imaging methods and AFP levels was applied in four cases (12.9%). All 31 patients presented
up to three liver nodules smaller than 3 cm in total. Some nodules were detected as part of a screening
program (55%) involving abdominal US and serum AFP monitoring every 6 months, while some were
referrals from other centers with diagnoses of suspected HCC. The mean nodule size was 22 mm. All
patients underwent a chest computerized tomography scan and a full-body bone scan to exclude the
presence of metastatic HCC.

Control group
Sixty-two patients (40 male, 22 female) with hepatitis C-related cirrhosis, but without HCC were selected
from the same tertiary care center. They were paired by age and gender with the HCC group. All patients in
the control group were subjected to abdominal US 6 months after data collection, to ensure that HCC had
not developed. These patients were systematically screened every 6 months for HCC with US and serum
AFP measurements.

The following anthropometric and clinical variables were recorded and used to categorize the control group:
age (> 60 years); gender (male/female); treatment with alpha-interferon (yes/no); previous participation in a
screening program (yes/no); response to antiviral treatment (yes/no); Child-Pugh score (A/B/C); esophageal
varices (yes/no); upper gastrointestinal (GI) bleeding (yes/no); ascites (yes/no); hepatic encephalopathy (yes/
no); spontaneous bacterial peritonitis (SBP) (yes/no); weight loss (yes/no); alcohol consumption (yes/no) and
abdominal pain (yes/no).

The following serum markers were examined: AFP (≥ 20 ng/mL), total bilirubin (Bil) (> 10 ng/dL), aspartate
aminotransferase (AST) (> 41 U/L), alanine aminotransferase (ALT) (> 37 U/L), alkaline phosphatase
(AP) (> 129 U/L), gamma-glutamyl transpeptidase (GGT) (> 61 U/L), transferin saturation (> 40%), ferritin
3
(> 150 ng/mL), international normalized ratio (INR) (> 1.20), platelet count (< 100,000/mm ), albumin
(< 3.4 g/dL), fibrinogen (< 150 mg/dL), glycemia (> 110 mg/dL). We additionally recorded a descriptive
analysis of the HCC histological type as well-, moderately- or poorly-differentiated. Of the 12 histologically
confirmed tumors, 11 were moderately-differentiated, and only 1 was well-differentiated, while none were
poorly differentiated.

This study was approved by the Institutional Review Board, fulfilling all of the requirements for retrospective
studies in human subjects, according to the guidelines of the 1975 Helsinki Declaration.

Statistical analysis
Quantitative variables are presented as median, first quartile and third quartile, and qualitative variables as
percentages. Differences between groups (presence/absence of HCC) regarding continuous variables were
verified via the Mann-Whitney test and association between categorized variables were checked by Fisher’s
test. P-values smaller than 0.05 were considered statistically significant.

Receiver operator curve (ROC) curve was applied to all continuous variables, and cutoff values were selected
[22]
to maximize the Youden index (MaxSe and MaxSp) . Simple and multivariable logistic regressions were
[23]
performed to predict HCC presence. Akaike Information Criterion (AIC) was used to select the most
informative variables in the backward strategy. Patients with missing data in a specific variable were
excluded from the analysis of that variable.

Finally, linear predictors from multiple regressions were resized to a range from 0 to 100, and then a cutoff
value was determined by a ROC curve. Performance measures given by sensitivity (Se), specificity (Sp),